Ethanol has multiple effects on DNA synthesis in fibroblasts depending on the presence of secreted growth regulators and zinc as well as the level of protein kinase C activation.

Earlier we showed that in serum-starved (27 h), washed mouse fibroblasts and other cell lines 40-80 mM concentrations of ethanol (EtOH) potentiate, in a zinc (Zn2+)-dependent manner, the combined stimulatory effects of calcium (Ca2+) and insulin (Ins) on DNA synthesis. We now report that the promitogenic EtOH effects require removal of the used medium at least 6 h prior to treatments with EtOH, Zn2+, and Ins. If serum-starved (27 h) cells were continuously incubated for another 18-h period without replacing the medium, a secreted cellular factor moderately enhanced the mitogenic effect of Ins and simultaneously blocked the potentiating effect of EtOH on DNA synthesis measured during the last hour of treatments. However, the presence of Ca2+ (2.8 mM) plus Zn2+ (25 microM) or 25-300 nM phorbol 12-myristate 13-acetate (PMA) during the serum starvation period partially restored the promitogenic effect of EtOH. The PMA effect was blocked by the protein kinase C (PKC) inhibitor GF 109203X added for the second (18 h) period. Even at 300 nM, PMA failed to fully downregulate PKC-alpha, the major PKC isoform, over a 28-h period, suggesting that an activated PKC enzyme was involved in the restoration of EtOH effect. When EtOH (40-80 mM) was added for the entire serum starvation period and the incubations were continued for 18 h without removing the medium, EtOH inhibited both the combined actions of Ins and cellular factor as well as the promoting effect of newly added EtOH on Ins-dependent DNA synthesis. Coaddition of Zn2+ and PMA with EtOH prevented these inhibitory effects of EtOH. The results indicate that in mouse fibroblasts EtOH can both enhance and inhibit Ins-dependent DNA synthesis depending on the timing of EtOH treatment as well as the presence of Zn2+, cellular factors, and activators of the PKC system.

Ethanol enhances the stimulatory effects of lysophosphatidic acid on DNA synthesis but not cell proliferation in human and mouse fibroblasts.

Lysophosphatidic acid (LPA), a constituent of serum, is a positive regulator of cell growth, while ethanol (EtOH) has been shown to exert both inhibitory and stimulatory effects on mitogenesis. In this work, we examined possible interactions between the effects of EtOH and LPA on DNA synthesis, cell proliferation, activating phosphorylation of p44/p42 mitogen-activated protein kinases (MAPK), and p70 S6 kinase (p70 S6K) activity. In fibroblasts derived from human or mouse embryo or the skin of healthy human subjects, LPA (1-20 microM) and EtOH (40-80 mM) synergistically stimulated DNA synthesis in a zinc-dependent manner. Nevertheless, EtOH did not modify the stimulatory effect of LPA on the proliferation of human embryonal fibroblasts. In the presence of zinc, EtOH did not affect LPA-induced activating phosphorylation of p42/p44 MAPKs, although an inhibitor of MAPK kinase inhibited the combined effects of LPA and EtOH on DNA synthesis. In contrast, in the presence of zinc, EtOH enhanced the stimulatory effect of LPA on p70 S6K activity. The results indicate that in human fibroblasts, in the presence of zinc, EtOH enhances the stimulatory effects of LPA on DNA synthesis, but not on cell proliferation, by a mechanism probably involving activation of p70 S6K.

Promitogenic effects of ethanol, methanol, and ethanolamine in insulin-treated fibroblasts.

The zinc-dependent potentiating effect of ethanol (EtOH) on insulin-stimulated DNA synthesis was studied with a focus on the possible site of EtOH action and the ability of other alcohols to elicit similar promitogenic effects. In serum-starved (27 hr) NIH 3T3 fibroblasts, 200-300 mM methanol (MeOH) and 0.1-1.5 mM ethanolamine (Etn), but not 3- to 9-carbon normal alcohols, enhanced the effect of insulin on DNA synthesis to varying extents. The promitogenic effects of EtOH and MeOH, but not that of Etn, required the presence of 15-25 microM zinc. The potentiating effects of Etn were enhanced by 5 mM choline (Cho) and inhibited by 1-3 mM hemicholinium-3 (HC-3), an inhibitor of Cho transporter and Cho kinase. In the presence of 15 microM zinc, 40 mM EtOH, which had no effect on its own, inhibited the potentiating effects of Cho and enhanced the inhibitory effects of HC-3 on synergistic stimulation of DNA synthesis by Etn and insulin. On the other hand, both Cho and HC-3 partially inhibited the promitogenic effect of 80 mM EtOH in the presence of 25 microM zinc. After a 10-min incubation, EtOH decreased the amount of cell-associated [(14)C]Cho in the absence but not in the presence of HC-3. After a 40-min incubation, Cho (5 mM) partially inhibited the cellular uptake as well as the metabolism of [(14)C]Etn. Whereas after the 40-min incubation 80 mM EtOH had no effects on Etn metabolism, in the absence of Cho it decreased the amount of cell-associated [(14)C]Etn. However, EtOH had no detectable effects on cell association of [(14)C]Etn after the 10-min incubation. The results suggest that in NIH 3T3 fibroblasts EtOH is a remarkably specific promitogen, and that it may act via a cell membrane site(s), also regulated by Cho (agonist) and HC-3 (antagonist), which can influence membrane binding and the promitogenic activity of Etn.

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

alpha(1)-antitrypsin can increase insulin-induced mitogenesis in various fibroblast and epithelial cell lines.

alpha(1)-Antitrypsin (AT), the archetypal member of the superfamily of serine proteinase inhibitors, inhibits leukocyte elastase activity and thereby can prevent lung damage. Here we show that in fibroblasts from human fetal lung and mouse embryo as well as in certain epithelial cells AT can also enhance the stimulatory effects of insulin on DNA synthesis and cell proliferation. Warming of AT at a moderate (41 degrees C) temperature for a longer time (21 h) or at a higher (65 degrees C) temperature for 30 min before treatment increased its stimulatory effects on both DNA synthesis and activating phosphorylation of p42/p44 mitogen-activated protein kinases. The results suggest that AT may promote regeneration of damaged tissues under pathophysiological conditions which are associated with fever.

Growth factor-like effects of placental alkaline phosphatase in human fetus and mouse embryo fibroblasts.

Human placental alkaline phosphatase (PALP) is synthesized in the placenta during pregnancy and is also expressed in many cancer patients; however, its physiological role is unknown. Here we show that in human fetus fibroblasts as well as normal and H-ras-transformed mouse embryo fibroblasts PALP stimulates DNA synthesis and cell proliferation in synergism with insulin, zinc and calcium. The mitogenic effects of PALP are associated with the activation of c-Raf-1, p42/p44 mitogen-activated protein kinases, p70 S6 kinase, Akt/PKB kinase and phosphatidylinositol 3'-kinase. The results suggest that in vivo PALP may promote fetus development as well as the growth of cancer cells which express oncogenic Ras.

Ethanol, Zn2+ and insulin interact as progression factors to enhance DNA synthesis synergistically in the presence of Ca2+ and other cell cycle initiators in fibroblasts.

In serum-starved NIH 3T3 fibroblasts, ethanol (30-80 mM) promoted the effects of insulin and insulin-like growth factor I (IGF-I) on DNA synthesis in a Zn(2+)-dependent manner. Ethanol and Zn(2+) were most effective when added shortly before or after insulin, indicating that all these agents facilitated cell cycle progression. The synergistic effects of ethanol, Zn(2+) and insulin (or IGF-I) on DNA synthesis required 1.1-2.3 mM Ca(2+), which seemed to act as the cell cycle initiator. When serum-starved cells were pretreated for 2 h with other cell cycle initiators such as 10% (v/v) serum, 50 ng/ml platelet-derived growth factor or 2 ng/ml fibroblast growth factor, subsequent co-treatments with 60 mM ethanol, Zn(2+) and insulin for an 18 h period again synergistically increased DNA synthesis. Of the various signal transducing events examined, ethanol stimulated cellular uptake of (45)Ca and it enhanced the stimulatory effects of insulin on p70 S6 kinase activity in a Zn(2+)-dependent manner. In contrast, ethanol inhibited insulin-induced activating phosphorylation of p42/p44 mitogen-activated protein kinases; these inhibitory ethanol effects were prevented by Zn(2+). The results show that, in NIH 3T3 fibroblasts, ethanol can promote cell cycle progression in the presence of a cell cycle initiator as well as Zn(2+) and insulin (or IGF-I).

Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.

In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.

The possible mechanism of synergistic effects of ethanol, zinc and insulin on DNA synthesis in NIH 3T3 fibroblasts.

In serum-starved NIH 3T3 fibroblast cultures, zinc (15-40 microM) enhanced both the individual and combined stimulatory effects of insulin and ethanol (EtOH) on DNA synthesis. Zinc, but not EtOH, also promoted the stimulatory effects of insulin on activating phosphorylation of p42/p44 mitogen-activated protein (MAP) kinases. In the presence of zinc, insulin induced premature expression of cyclin E during early G1 phase; EtOH partially restored the normal timing (late G1 phase) of cyclin E expression. The results suggest that zinc and EtOH promote insulin-induced DNA synthesis by different mechanisms; while zinc acts by enhancing the effects of insulin on MAP kinase activation, EtOH may act by ensuring timely zinc-dependent insulin-induced expression of cyclin E.

Potentiation of calcium-mediated stimulation of DNA synthesis by ethanol in human and mouse fibroblasts.

Alcohol abuse is a risk factor for cancers of the gastrointestinal tract, and it also can precipitate psoriasis characterized by hyperproliferation of epidermal cells. Because these effects of alcohol may involve stimulation of cell growth, and ethanol (EtOH) was shown to enhance DNA synthesis in mouse fibroblasts and epidermal cells, we conducted a study to determine whether EtOH can also stimulate mitogenesis in human fibroblasts and keratinocytes. In keratinocytes, EtOH had no effects on mitogenesis after shorter (17-hr) treatments, but it partially prevented inhibition of DNA synthesis elicited by longer treatments (3-4 days) with 2 mM calcium (Ca2+), a differentiation-inducing agent. In contrast, treatment of serum-starved zinc-treated (40 microM) human skin fibroblasts with 50-60 mM EtOH for 17 hr resulted in increased DNA synthesis. EtOH-induced DNA synthesis was blocked by 1 mM EGTA, a specific Ca2+ chelator. Despite the presence of 1.8 mM Ca2+ in the cell culture medium, the addition of 1 mM extra Ca2+ (final concentration, 2.8 mM) for 17 hr induced DNA synthesis, presumably mediated by Ca2+ receptors. In eight independent human skin fibroblast lines examined, treatment with EtOH for 46 hr, but not for 17 hr, invariably enhanced the effects of Ca2+ on DNA synthesis, consistent with synergistic stimulation of cell proliferation by EtOH and Ca2+. Neomycin, a Ca2+ receptor agonist, and EtOH also exerted synergistic effects on DNA synthesis after longer (46-hr) treatments. In mouse NIH 3T3 fibroblasts, both EtOH- and Ca2+-enhanced DNA synthesis after 17-hr treatment, but they stimulated cell proliferation only in combination. The results indicate that in human fibroblasts, EtOH can potentiate the longer-term effects of high concentrations of Ca2+ on DNA synthesis whereas, in keratinocytes, EtOH may inhibit Ca2+-induced differentiation.

Phorbol ester stimulation of phosphatidylcholine synthesis requires expression of both protein kinase C-alpha and phospholipase D.

The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates both the synthesis and phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho). Here, attached and suspended NIH 3T3 fibroblasts as well as variants of the MCF-7 human breast carcinoma cell line expressing PKC-alpha and a PtdCho-specific PLD activity at widely different levels were used to determine the possible role of PKC-alpha, PtdCho hydrolysis, and choline uptake in the mediation of PMA effect on PtdCho synthesis. In wild-type MCF-7 cells, which express both PKC-alpha and PLD activities at very low levels, PMA had little effects on the uptake or incorporation [14C]choline into PtdCho. In multidrug resistant MCF-7/MDR1 cells, which highly express PKC-alpha but lack the PtdCho-specific PLD activity, 100-nM PMA had relatively small stimulatory effects on the uptake of [14C]choline (approximately 1.5-fold) and [14C]PtdCho synthesis (1.5- to 2-fold). In NIH 3T3 fibroblasts and MCF-7/PKC-alpha cells, both expressing PKC-alpha and PLD activities at high levels, 10-100-nM PMA enhanced [14C]choline uptake only slightly (1.7- to 2.2-fold), while it had much greater (approximately 4-9-fold) stimulatory effects on PtdCho synthesis. PMA significantly enhanced the formation of phosphatidic acid (PtdOH) in MCF-7/PKC-alpha cells (2.8-fold increase), but not in MCF-7/MDR1 cells (1.4-fold increase), while in both cell lines it had only small (1.3-1.5-fold) stimulatory effects on 1,2-diacylglycerol (1, 2-DAG) formation. In suspended NIH 3T3 cells, 200-300-mM ethanol blocked the stimulatory effect of PMA on PtdOH formation without affecting PtdCho synthesis indicating that neither PtdOH nor 1,2-DAG derived from it is a mediator of PMA effect on PtdCho synthesis. In attached NIH 3T3 cells, dimethylbenz[a]anthracene enhanced phosphocholine formation and, thus, choline uptake without increasing PtdCho synthesis or modifying the effect of PMA. While the results indicate that the stimulatory effect of PMA on PtdCho synthesis requires the expression of both PKC-alpha and a PtdCho-specific PLD, they do not support a role for 1,2-DAG, PtdOH or choline in the mediation of PMA effect.

The choline kinase inhibitor hemicholinium-3 can inhibit mitogen-induced DNA synthesis independent of its effect on phosphocholine formation.

In NIH 3T3 cells, phosphocholine (PCho) stimulates mitogenesis in synergism with insulin, ATP, and sphingosine-1-phosphate (S1P) via an extracellular target. Intracellular PCho also has been suggested to mediate the mitogenic effects of fibroblast growth factor (FGF) and several other growth factors based, in part, on the observed inhibition of growth factor-induced mitogenesis by the choline kinase inhibitor hemicholinium-3 (HC-3). Here we examined the specificity of HC-3 effects on mitogenesis in serum-starved NIH 3T3 and Swiss 3T3 cells. In both cell lines, FGF greatly enhanced DNA synthesis in a medium containing 28 microM choline, and it also stimulated the formation of -14C-PCho from both 50 microM and 5 mM [14C]choline. HC-3 (2 mM) inhibited basal or FGF-induced formation of [14C]PCho and [14C]phosphatidylcholine as well as the uptake of -14C-choline only at the 50 microM, but not the 5 mM, concentration of [14C]choline. In addition, HC-3 (1 mM) from three different sources (95-99.9% purity) inhibited FGF-stimulated DNA synthesis by 53-58% which was not reversed by 5 mM choline. The choline analogue dimethylethanolamine (1 mM) also inhibited FGF-stimulated formation of [14C]PCho from 50 microM -14C-choline, but it had no effect on FGF-induced DNA synthesis. Of the other growth regulators examined, synergistic stimulation of DNA synthesis by extracellular PCho and S1P or PCho and ATP via choline kinase-independent mechanisms was inhibited by 2 mM HC-3. However, HC-3 failed to inhibit the synergistic mitogenic effects of PCho and insulin or S1P and insulin. The results suggest that FGF-induced mitogenesis does not require PCho formation and that HC-3 can inhibit DNA synthesis independent of its inhibitory effects on choline metabolism.

Phosphorylation of ethanolamine, methylethanolamine, and dimethylethanolamine by overexpressed ethanolamine kinase in NIH 3T3 cells decreases the co-mitogenic effects of ethanolamines and promotes cell survival.

Ethanolamine (Etn), as well as its N-methyl (MeEtn) and N,N-dimethyl (Me2Etn) analogues, were recently shown to potentiate the stimulatory effect of insulin on DNA synthesis in serum-starved NIH 3T3 fibroblasts. In the present work we assessed the impact of the co-mitogenic effects of Etn and its methyl analogues on cell proliferation and cell survival, and examined whether the cell growth regulatory effects of these ethanolamines involve an Etn-kinase-mediated phosphorylation step. For this purpose, NIH 3T3 sublines highly overexpressing Drosophila Etn kinase and an appropriate vector control line were utilized and the effects of Etn, MeEtn, Me2Etn, methylamine (MeNH2), and dimethylamine (Me2NH) were studied. 31P-NMR analysis of the water-soluble cell metabolites revealed that both MeEtn and Me2Etn, but not choline, are excellent substrates for the expressed Etn kinase. The methylated ethanolamines (MeEtn and Me2Etn) and methylamines (MeNH2, Me2NH) were used as Etn models that can or cannot be phosphorylated, respectively. In serum-starved vector control cells, both MeNH2 (1 mM) and Me2NH (1 mM) were more effective than Etn in enhancing insulin-induced DNA synthesis, and both were almost as effective as MeEtn and Me2Etn. However, in the Etn kinase overexpressor cells the potentiating effects of Etn, MeEtn and Me2Etn, but not those of MeNH2 and Me2NH, were significantly reduced. Moreover, in the overexpressor cells, lower concentrations of Etn (50-200 microM) inhibited the combined mitogenic effects of Me2NH (1 mM) and insulin. These data are consistent with a mechanism in which the phosphorylated and non-phosphorylated ethanolamines are negative and positive regulators of insulin-induced mitogenesis, respectively. After incubating the cells for 13 days in serum-free medium in 96-well microplates, there was a steady decrease in cell numbers in both cell lines. However, between 6-13 days, 0.1-1 mM MeEtn and, particularly, Me2Etn provided significant protection against cell death in the Etn kinase overexpressor cells. In vector control cells, only Me2Etn in combination with insulin had similar effects on cell survival. The data suggest that phosphorylated ethanolamines may function as promoters of cell survival.

Stimulation of DNA synthesis in untransformed cells by the antiviral and antitumoral compound tricyclodecan-9-yl-xanthogenate (D609).

The antiviral and antitumor xanthate compound tricyclodecan-9-yl-xanthogenate (D609) is best known for its inhibitory effect on phosphatidylcholine-specific phospholipase C activity. Now we report that in NIH 3T3 cells, but not in several transformed cell types tested, D609 stimulated DNA synthesis when phosphocholine (PCho), insulin, or ATP was also present. Maximal co-mitogenic effects of D609 were observed at 5 microg/mL, a concentration 4-6 times lower than that required to inhibit phospholipase C activity. The synergistic mitogenic effects of D609 and PCho, but not of D609 and insulin, were associated with activation of p42 and, to a lesser extent, p44 mitogen-activated protein (MAP) kinases. The results raise the possibility that the mitogenic activity of D609 in untransformed cells may contribute to its antiviral and antitumor effects.

Bombesin and zinc enhance the synergistic mitogenic effects of insulin and phosphocholine by a MAP kinase-dependent mechanism in Swiss 3T3 cells.

Simultaneous treatment of serum-starved (24 h) Swiss 3T3 cells with insulin (500 nM) and phosphocholine (PCho) (0.25-1 mM) resulted in synergistic stimulation of DNA synthesis via a mitogen activated protein (MAP) kinase-independent rapamycin-sensitive mechanism. Co-treatment of cells with bombesin (10 nM) or zinc (25 microM) enhanced the combined mitogenic effects of insulin and PCho 2-3-fold; however, in the presence of bombesin or zinc the combined effects of insulin and PCho were not inhibited by rapamycin. The potentiating effects of bombesin and zinc on insulin plus PCho-induced DNA synthesis were accompanied by large stimulation of p42 MAP kinase activity. The results indicate that in Swiss 3T3 cell cultures, synergistic stimulation of DNA synthesis by extracellular insulin and PCho via a p42 MAP kinase-dependent mechanism requires the presence of other growth regulatory agents, such as bombesin or zinc.

Alkyl lysophospholipids inhibit phorbol ester-stimulated phospholipase D activity and DNA synthesis in fibroblasts.

The antineoplastic alkyl lysophospholipids (ALP) 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) and 1-S-hexadecylthio-2-methoxymethyl-2-deoxy-rac-glycero-3-phosphocho line (BM41.440) were found to alter phospholipase D (PLD)-mediated phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) hydrolysis in NIH 3T3 fibroblasts. After a shorter (50 min) treatment, 2.5-7.5 microg/ml concentrations of ALP stimulated PtdCho, but not PtdEtn, hydrolysis 2-4-fold. At the same time, 7.5-25 microg/ml concentrations of ALP significantly inhibited the larger (5.8-6.5-fold) stimulatory effects of phorbol 12-myristate 13-acetate (PMA) on both PtdCho and PtdEtn hydrolysis. When a brief (30 min) exposure of cells to 1-2.5 microg/ml concentrations of BM 41.440 was followed by incubation of washed cells for 3-16 h prior to the assay of PLD activity or DNA synthesis, the treated cells exhibited no increased PtdCho hydrolysis, while their responses to the stimulatory PMA effects on both PLD activity and DNA synthesis were strongly reduced. The results suggest that the PLD and protein kinase C systems may be important cellular targets of ALP actions.

ATP-dependent choline phosphate-induced mitogenesis in fibroblasts involves activation of pp70 S6 kinase and phosphatidylinositol 3'-kinase through an extracellular site. Synergistic mitogenic effects of choline phosphate and sphingosine 1-phosphate.

In serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0.5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0.1-1 mM) both in the absence and presence of insulin; maximal potentiating effects required 50-100 microM ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10-100 microM) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3'-kinase (PI 3'-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3'-kinase or treatments with the PI 3'-kinase inhibitor wortmannin only partially (approximately 40-50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3'-kinase.

Extracellular sphingosine 1-phosphate stimulates formation of ethanolamine from phosphatidylethanolamine: modulation of sphingosine 1-phosphate-induced mitogenesis by ethanolamine.

In this work, we determined the effects of sphingosine 1-phosphate (S1P) on phospholipase D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn), and evaluated the effects of the water-soluble product ethanolamine on S1P-induced DNA synthesis in NIH 3T3 cells. In [14C]ethanolamine-labelled cells, S1P (0.5-5 microM) stimulated PLD-mediated hydrolysis of PtdEtn 1.5-2.1-fold. Down-regulation of protein kinase C by chronic (24 h) treatment of cells with 300 nM PMA, or pretreatments (10 min) with the cell-permeant calcium chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N, N',N'-tetra-acetic acid tetra-acetoxymethyl ester led to the inhibition of S1P-induced PtdEtn hydrolysis. S1P alone was a weak inducer of DNA synthesis, but its effects were enhanced by phosphocholine (PCho), insulin, ATP or PMA. Ethanolamine (5-100 microM) did not modify the mitogenic effect of S1P alone, whereas at 50-100 microM concentrations it actually enhanced the mitogenic effect of PCho via a mitogen-activated protein (MAP) kinase-independent mechanism. In contrast, 5-20 microM concentrations of ethanolamine, which correspond to normal blood ethanolamine levels in humans, strongly inhibited DNA synthesis induced by S1P plus PCho via a MAP kinase-dependent mechanism; importantly, less or no inhibition was observed with 50-100 microM concentrations of ethanolamine. At 5-50 microM concentrations, ethanolamine also inhibited the synergistic mitogenic effects of both S1P plus insulin (22-27% inhibition) and PCho plus ATP (45-73% inhibition) but not those of S1P plus PMA or S1P plus ATP. The results indicate that S1P stimulates PLD-mediated hydrolysis of PtdEtn by a mechanism that may involve a regulatory protein kinase C isoform. Increased formation of ethanolamine by PLD-mediated PtdEtn hydrolysis or by other means may be required for maximal stimulation of DNA synthesis by S1P in the presence of insulin, and particularly PCho.

Ethanolamine, but not phosphoethanolamine, potentiates the effects of insulin, phosphocholine, and ATP on DNA synthesis in NIH 3T3 cells--role of mitogen-activated protein-kinase-dependent and protein-kinase-independent mechanisms.

NIH 3T3 fibroblasts express a phospholipase D activity hydrolyzing phosphatidylethanolamine (PtdEtn) which produces ethanolamine (Etn) in response to a variety of growth regulating agents. The main objective of this work was to evaluate the effects of Etn on mitogenesis and to determine whether these effects require its metabolism to phosphoethanolamine (PEtn) or PtdEtn. To increase conversion of Etn to PEtn, an Etn-specific kinase derived from Drosophila was highly expressed in NIH 3T3 cells. Overexpression of this Etn kinase resulted in large (10-12.5-fold) increases in PEtn formation, but only in modest (1.2-1.7-fold) increases in PtdEtn synthesis. In both vector control and Etn kinase overexpressor cells, Etn had biphasic effects on insulin-induced DNA synthesis with maximal (approximately 2-fold) potentiating effects being observed at 0.5-1 mM concentrations, followed by an inhibitory phase at higher Etn concentrations. In the Etn kinase overexpressor lines, the inhibitory phase was elicited by lower Etn concentrations and it was partially blocked by 5 mM choline due to decreased formation of PEtn. In both vector control and Etn kinase overexpressor cells, phosphocholine (PCho) and insulin synergistically stimulated DNA synthesis; their effects were further enhanced by physiologically relevant (5-60 microM) concentrations of Etn by a mechanism independent of mitogen-activated protein (MAP) kinase. Concentrations of Etn >50 microM also enhanced the effects of both PCho and the synergistic effects of PCho plus ATP; however, in the latter case 20 microM Etn was inhibitory. The magnitude of both the potentiating and inhibitory effects of Etn on PCho-induced as well as PCho + ATP-induced DNA synthesis were similar in the vector control and Etn kinase overexpressor cells; they were associated with stimulation and inhibition, respectively, of p42 MAP kinase activity. The results indicate that in NIH 3T3 cells Etn exerts significant effects on DNA synthesis which, except inhibition of insulin-induced DNA synthesis by higher concentrations of Etn, do not correlate with the metabolism of Etn to PEtn or PtdEtn.

Protein kinase C inhibitors enhance the synergistic mitogenic effects of ethanolamine analogues and insulin in NIH 3T3 fibroblasts.

Monomethylethanolamine (1 mM) and dimethylethanolamine (1 mM) stimulated DNA synthesis 10- and 15-fold, respectively, in NIH 3T3 fibroblasts. In addition, simultaneous treatments with insulin (500 nM) and methylated ethanolamine analogues (1 mM or less) resulted in synergistic activation of DNA synthesis. The order of mitogenic potency of ethanolamine analogues was dimethylethanolamine > monomethylethanolamine > ethanolamine. Choline (1-5 mM) alone had no effect on DNA synthesis, but it increased the combined effects of lower concentrations of ethanolamine analogues and insulin. The synergistic effects of ethanolamine analogues, choline and insulin were considerably (1.7- to 1.9-fold) enhanced by GF 109203X (3 microM), a specific inhibitor of protein kinase C. The results suggest that ethanolamine analogues enhance insulin-induced DNA synthesis by a mechanism which is inhibited by the protein kinase C system.